The objective of the proposed work is to determine and characterize the spatio-temporal limits of the metabolic and hemodynamic responses that result from evoked neural activity. For this purpose a genetically enhanced mouse model expressing the light-sensitive protein Channelrhodopsin-2 will be used whereby the neuronal activity can be controlled through the modulation of the diameter and intensity of a light stimulus delivered directly onto the surface of the brain (photo-stimulus). First, the minimum neuronal activity that elicits a blood flow response will be determined using two-photon microscopy with sub-micrometer resolution. The size and strength of the neuronal activity will be characterized using a calcium indicator. Then, an intra-vascular fluorescent dye will be used to measure corresponding changes in blood velocity and vessel diameter in capillaries. Second, the consistency of these findings and the effect of resolution will be investigated by measuring the functional cerebral blood flow, metabolic and overall hemodynamic responses using larger- scale optical imaging with resolution of tens of micrometers. Specifically, the following optical methods will be used to measure blood flow, metabolic and overall hemodynamic responses to neural activity: laser speckle imaging, intrinsic auto-fluorescence imaging and optical imaging of intrinsic signal, respectively. The latter is a direct analog of blood oxygenation level dependent functional magnetic resonance imaging (BOLD fMRI). As a result of this project, the spatio-temporal limits of the metabolic and hemodynamic responses will be uncovered with unparalleled resolution. In addition, sufficient knowledge will be obtained to elucidate the theoretical and practical requirements of current brain research tools like fMRI to detect activities encoded in the hemodynamic response that may be thought to be undetectable. This work will have a tremendous impact in brain imaging studies of function in animals and humans, including clinical and basic science research. To perform the proposed studies I will undergo training in two-photon imaging of brain function and complementary optical imaging methods, and in electrophysiological recording of neuronal activity. A unique combination of local and national experts will provide and oversee the necessary training. This proposal is vital in fulfilling my ultimate career goal of attaining an independent and successful research program that focuses on imaging normal and pathological brain function. PUBLIC HEALTH RELEVANCE: The hemodynamic response induced by neural activity is, and has been, the principal means of studying brain function in animals and humans. Methods like functional magnetic resonance imaging are now essential in basic science, developmental, cognitive and clinical studies of normal and impaired brain function. This proposal will reveal the fundamental limits of hemodynamic-based methods to image and detect brain function.